EP0749498B1 - Combined use of genetic markers for the diagnosis of alzheimer's disease, diagnostic kit and method - Google Patents

Abstract

PCT No. PCT/FR95/00259 Sec. 371 Date Jan. 2, 1997 Sec. 102(e) Date Jan. 2, 1997 PCT Filed Mar. 6, 1995 PCT Pub. No. WO95/24504 PCT Pub. Date Sep. 15, 1995Combined use of at least two genetic markers selected from apolipoprotein E, D19S178 and apolipoprotein CII, for the diagnosis of Alzheimer's disease, especially apolipoprotein epsilon 4, long apolipoprotein CII (30+/-3 repeat patterns (CA) and short D19S178 (less than 167+/-4 nucleotides) alleles. The invention also concerns a method for the diagnosis of Alzheimer's disease and a kit for carrying out said method.

Description

The present invention relates to markers genetics used jointly for the diagnosis of Alzheimer's disease.

Alzheimer's disease is a pathology encephalic characterized by early dementia with loss of cortical neurons associated with plaques of β-amyloid, tangles of neurofibrils and in most cases amyloid angiopathy. he there are strong presumptions for genetic influence in the etiology of Alzheimer's disease (WO 94/01772).

This genetic component has been highlighted for many years by observations indirect which suggest a mode inheritance autosomal dominant and penetrance dependent on age to explain the family ties between individuals affected by the disease. Genetic studies molecular research have isolated genes putative of Alzheimer's disease by the search for chromosome specific polymorphic genetic markers (Bird et al., 1989, Neurobiology of Aging 10, 432-434).

Three chromosomal locations were described as being involved in family forms with early onset (age of onset less than 60 years): chromosome 21, chromosome 14 and chromosome 19. Two linkage studies have suggested that the region chromosome 19q13.2 was associated with forms of late onset Alzheimer's disease (Pericak-Vance et al, Am. J. Hum. Broom. (1991), 48, 1034-1050 ; Schellenberg et al., Ann. Neurol. (1992), 31, 223-227). Within this chromosomal region, the group of apolipoprotein (APO) genes E-CI-CI'-CII is a candidate area. Among the products of these genes, apolipoprotein E (APOE) is particularly involved in the nervous system: APOE is present in plaques senile and has a bonding affinity with the β-A4 peptide. Strittmatter et al. (Proc. Natl. Acad. Sci. (1993) 90, 177-181) described a frequency increased by the ε4 allele of the APOE gene in the forms late onset Alzheimer's disease. This observation has been confirmed for the forms family (Corder et al., Science (1993), 261, 921-923) and sporadic forms of Alzheimer's disease (Corder et al., Science (1993), 261, 921-923; Saunders et al., Neurology - (1993), 13, 1467-1472).

On the other hand, Schellenberg et al. (Ann. Neurol. 1992, 31: 223-227) reported an association genetics between the F allele of the apolipoprotein gene CII (allele of the length polymorphism of fractions of restriction of TaqI (RFLP: Restriction Fragment Length Polymorphism)) and the familial form of Alzheimer's disease.

The authors of the present invention have carried out a study on two populations of origin different attacks one of a form occurred late Alzheimer's disease (after 65 years), the other of an early-onset form of Alzheimer's disease (before age 65), which has made it possible to establish an increase significant in both groups of at least two of following genetic markers: APOE ε4 allele, allele D19S178 short and allele APO CII long.

The localization on chromosome 19 of the markers APOE, APO CII (APO C2) and D19S178 is known and described in particular by Williamson et al. (Cyto Genetic and Cell Genetic 1991, vol. 58 , p. 1678).

The object of the present invention is thus the joint use of at least two markers genetics chosen from APOE, D19S178 and APO CII for diagnosis of Alzheimer's disease, markers preferably being made up of APOE and D19S178 and / or APO CII, more preferably by APOE, D19S178 and APO CII.

The genetic markers used are advantageously the APOE ε4 allele, the D19S178 allele short and the long APO CII allele.

The APOE gene has three alleles: ε2, ε3 and ε4.

By long APO CII allele, we mean an allele comprising more than 30 ± 3, preferably more than 30 consecutive repetitions of cytosine-adenine bases.

By short D19S178 allele means an allele comprising less than 167 ± 4, preferably less than 167 nucleotides.

The invention also relates to a method for diagnosing Alzheimer's disease characterized by what we look for in a biological sample of a patient the presence of at least two markers following: APOE ε4 allele, short D19S178 allele and allele APO CII long.

a) mise en contact de l'échantillon biologique contenant de l'ADN avec un couple d'amorces spécifiques permettant l'amplification de tout ou d'une partie des gènes APOE, D19S178 et/ou APO CII, l'ADN humain contenu dans l'échantillon ayant été éventuellement rendu accessible à l'hybridation et dans des conditions permettant une hybridation des amorces à l'ADN humain contenu dans l'échantillon biologique ;

b) amplification de l'ADN humain ;

c) révélation des produits d'amplification par les techniques appropriées ;

d) détection de la présence éventuelle des allèles APOE ε4, D19S178 court et APO CII long par les techniques appropriées.

The method according to the invention advantageously comprises the following steps:

a) bringing the biological sample containing DNA into contact with a pair of specific primers allowing the amplification of all or part of the APOE, D19S178 and / or APO CII genes, the human DNA contained in the sample having possibly been made accessible to hybridization and under conditions allowing hybridization of the primers to the human DNA contained in the biological sample;

b) amplification of human DNA;

c) revelation of the amplification products by appropriate techniques;

d) detection of the possible presence of the APOE ε4, short D19S178 and long APO CII alleles by appropriate techniques.

By diagnosis, within the meaning of this invention means confirmation of the presence of at at least two markers chosen from those described above in patients whose clinical picture reports symptomatology that can be attributed to the disease Alzheimer's, or an increased likelihood in subjects of developing Alzheimer's disease compared to an entire population, the increase in probability being at least a factor of 4.

The biological sample can be blood total, the leukocyte fraction of the blood or a tissue from which DNA can be extracted, or a biological fluid.

The specific primers used in the framework of the present invention may be in particular those described in Roppers et al. (1991), Cytogenet. Cell Genet. 58, 751-784) for D19S178, Hixson et al. (1990), J. Lipid. Res. 31, 545-548 for APOE and Weber and al., Am. J. Hum. Broom. 44, 388-396 for APO CII.

Other primers may be suitable, the criterion selection of primers being that they allow amplification of the parts of the APOE, D19S178 and APO CII with the respective polymorphisms: region 112-158 of APOE, 5 'end of APO CII.

The alleles sought, in particular D19S178 and APO CII long, can be highlighted by determination the length of the amplified fragments, for example by polyacrylamide gel electrophoresis or by determination of the sequence of the amplified fragment.

The alleles sought, in particular APO ε4, can also be detected by the analysis technique conformational polymorphism of single strands ("Single-Strand Conformation Polymorphism" (SSCP) such as described by Masato Orita et al., or by probe hybridization using a specific probe.

• des couples d'amorces spécifiques des gènes APOE, D19S178 et APO CII,
• les réactifs nécessaires pour effectuer une amplification d'ADN,
• éventuellement les réactifs permettant la détection des allèles APOE ε4, D19S178 court et/ou APO CII long, par exemple des réactifs nécessaires à une électrophorèse sur gel de polyacrylamide et la révélation des fragments après migration
• éventuellement des standards de référence constitués par les allèles sauvages des gènes mutants.

The present invention also relates to a kit for implementing the method according to the invention, in a sample comprising the following elements:

• pairs of primers specific to the APOE, D19S178 and APO CII genes,
• the reagents necessary to carry out DNA amplification,
• possibly the reagents allowing the detection of the APOE ε4, short D19S178 and / or APO CII long alleles, for example reagents necessary for an electrophoresis on polyacrylamide gel and the revelation of the fragments after migration
• possibly reference standards constituted by the wild alleles of the mutant genes.

The present invention also relates to amplification products of all or part of at least two genes chosen from APOE, APO CII and D19S178.

It also targets a composition of diagnosis made up of at least two markers genetics chosen from APOE, APO CII and D19S178.

The results of the study of the inventors who are authors of the present invention, showing the implication of the APOE, APO CII and D19S178 in the frequency of onset of the disease Alzheimer's.

The study focused on two groups of patients with Alzheimer's disease, a French group composed of 36 patients with a form to occur late Alzheimer's disease (78 ± 9 years; interval 65-91 years) and a British group made up of 34 patients with an early-onset form of Alzheimer's disease (57 ± 4 years; range: 49-64 years) diagnosed by the clinic.

These two groups were compared to two control groups of the same origin and age, composed 38 and 36 subjects respectively whose environment was identical to that of patients with Alzheimer's disease.

Patient genomic DNA was extracted from leukocytes according to the method described by Marcadet et al. (Standardized Southern-Blot Workshop Technique-Histocompatibility Testing, Springer Verlag, New York, Vol. 1). The genomic DNA was amplified by PCR using a Perkin Elmer Cetus amplification device.

Five markers located in the chromosomal region 19q13.2 were studied as described in the corresponding references: two anonymous markers containing motifs (CA) _n , D19S47 (referenced under Mfd 9 (Weber et al. (1989), Am. J. Hum. Genet. 44, 388-396)) and D19S178 (referenced under Mfd 139 (Ropers et al. (1991) Cytogenet. Cell Genet. 58, 751-784)), the APOE polymorphism (Hixson et al. (1990) J. Lipid. Res. 31, 545-548), restriction fragment length polymorphism (RFLP) for HpaI of the 5 'end of the Apo CI gene locus (Nillesen et al. (1990), Nucleic Acids Res . 18, 3428) and the polymorphism of repeating units (CA) _n in the APO CII gene (referenced under Mfd 5) (Weber et al., Am. J. Hum. Genet. 44, 388-396).

Statistical analyzes were performed with SAS software version 6.04. "Univariates" analyzes were performed using the Pearson χ ² test and the Fisher exact test if necessary. Given the rarity of the frequency of certain alleles observed for microsatellite polymorphisms, the alleles were grouped into two groups according to their number of nucleotides. Thus, D19S178 has been divided into long alleles (167 nucleotides and beyond) and short alleles (less than 167 nucleotides), the alleles of the 5 ′ end of APO CII with repeating units have been divided into long alleles (30 units repetitive and beyond) and short alleles (less than 30 repeating patterns).

Allelic frequencies have been calculated by counting alleles.

The collected results were processed by computer using a stepwise logistic regression model using statistical likelihood tests as described by Breslow NE et al., Statistical Methods in Cancer Research, Vol. 1, pp. 192-247. The analysis of the linkage imbalances was made as described by Thompson EA et al., Am. J. Hum. Genet., 42, 113-124 and Hill, WG, 1974, Hereditary, 33, 229-239.

The frequencies of the markers D19S178, APOE, APO CI and APO CII were estimated using the algorithm described by McLean and Morton, Genet. Epidemiol. 2, 263-272, put in the form of a computer program, as described by Cox et al., Am. J. Hum. Genet., 43, 495-501.

The estimated frequencies were compared to those expected based on the Pearson χ ² equilibrium or the Fischer exact test.

The results are reported in Tables I to IV below.

Table I reports the distributions of allelic frequency of genomic polymorphisms for sporadic subjects with Alzheimer's disease compared to witnesses.

No significant difference appears between subjects and witnesses in the distribution of the D19S47 allele for the different populations. In the late onset population, APOE and APO CII gene polymorphism frequencies were significantly different between subjects and controls: the APOE ε4 allele and the APO CII alleles were observed more frequently in subjects late onset of Alzheimer's disease than in witnesses.

The same analyzes were carried out for a independent UK population of sporadic subjects early onset compared to controls. This population was studied for the two age limits of generally accepted, i.e. less than 65 and under 60. For the group of subjects including age of onset was less than 65 years, distributions allelic polymorphisms of D19S178 genes, APOE and APOCI were significantly different between subjects and controls: short D19S178 alleles, the APOE ε4 allele and the presence of the restriction site of HpaI in APO CI (allele 2) were more frequently observed in patients where the onset of the disease was earlier than in controls. For groups of subjects with a lower age of onset of disease at 60, the allelic distributions of polymorphisms D19S178, APOE and APO CI genes were similar to those of the group of subjects with an age of onset of illness below 65 years.

For the following analyzes, the limit of age separation between the group at early onset and the late onset group was 65 years old.

The frequency observed for the APOE ε4 allele in the group of early-onset patients was not significantly different from that observed in the group of late-onset patients, and in both populations, the APOE ε2 allele was rare in subjects with Alzheimer's disease.

An inverse correlation between the age of occurred and the number of copies of the APOE ε4 allele was observed in the late onset population (p < 0.026), but not in the early-onset population. Average ages of onset for subjects with two alleles APO ε4, an ε4 allele or without the APO ε4 allele were 70.5, 75.0 and 81.2 years respectively in the group late onset, and 58.5, 56.6 and 57.3 years in the group with early onset.

The results of studies of imbalance of link two by two between the D19S178 polymorphisms, APOE, APO CI and APO CII are reported in Table II.

No deviation from the Hardy-Weinberg equilibrium was not observed. A link imbalance complete was found between the APO ε4 allele and the allele 2 of the restriction fragment length polymorphism (RFLP) of APO CI in the two populations.

In the control population, an imbalance of non-significant link was found between D19S178 and APOE polymorphisms and within the whole APOE-CI-CI'-CII.

The haplotypic frequencies of the 4 markers (D19S178, APOE, APO CI and APO CII) were estimated as reported in Table III below.

Among the 24 theoretical haplotypes, only 15 were found with the algorithm by the method described by McLean and Morton. Some haplotypes were observed only in patients (n ° 1, 2 and 7) and others only in controls (nos. 12 and 13) at the both in the late onset population and in the early onset. Haplotype # 10 was twice as large common in the late onset patient population than in the early-onset population.

The information content of polymorphism (Polymorphism Information Content (PIC)) and the degree of heterozygosity obtained by combining these 4 markers were raised in both groups.

The differences in the distribution of estimated haplotype frequencies between patients and controls were tested using the report ratio test probability (likelihood ratio test) with a number of degree of freedom equal to 15. The results were 88.4 and 93.2 respectively for the patient group at late onset and group of patients onset precocious.

To estimate the tendency to develop the disease ("odds ratio") for carriers of at least one alleles of different genotypes, we used stepwise multiple logistic regression models, which were adapted to the observations.

In the late-onset group, the "odds estimated ratios were 6.49 for the APOE ε4 allele (95% confidence interval = [1.68; 25.03]), 0.10 for the APOE 2 allele (95% confidence interval = [1.19; 10.70]).

In the early-onset group, the "odds estimated "ratios obtained were 3.80 for the APO allele ε4 (95% confidence interval = [0.01; 0.85]) and 4.44 for the short alleles of D19S178 (range of 95% confidence = [1.27; 15.49]).

The adjusted odds ratio for carriers of at least one APOE ε4 allele develop sporadic Alzheimer's disease type early onset or late onset was 4.10 (95% confidence interval = [1.84; 9.16]), like estimated in a regression model, adapted logistics for data relating to the total population.

For carriers of at least one allele, APOE ε2, the odds ratio was 0.11 (confidence interval 95% = [0.02; 0.50]), thus suggesting a protective effect of this allele.

The risk of developing Alzheimer's disease for carriers of at least one ε4 allele and minus one of the two markers: allele D19S178 short and APO CII long allele is reported in Table IV below.

In both populations, the odds risks ratios "were significantly increased when one of these two markers was considered at the same time as the APOE ε4 allele.

For example, the risk of reporting a late onset Alzheimer's disease for one subject carrying at least one APOE ε4 allele and at least one APO CII long allele is maximal, this configuration never found in the witnesses. In this same late-onset population, for carriers of at least at least one APOE ε4 allele and at least one D19S178 allele short, the odds ratio is 14 (14.23). This odds is also increased (greater than 8 (8.68)) for these same alleles in early Alzheimer's disease precocious.

In the general population, the risk of developing Alzheimer's disease regardless of the age of onset is increased for carriers of at least one APOE ε4 allele and at least one short D19S178 allele, as shown by l elevation of the odds ratio greater than 12 (12.46) as well as for the carriers of at least one APOE ε4 and at least one APO CII long allele (odds ratio greater than 9 (9.72)).

Claims (12)

1. Combined use of at least two genetic markers selected from APOE, D19S178 and APO CII for the diagnosis of Alzheimer's disease.
2. Use according to Claim 1 of the markers APOE, APO CII and/or D19S178.
3. Use according to Claim 1 of Claim 2 of the markers APOE, D19S178 and APO CII.
4. Use according to any one of the preceding claims, characterised in that the markers are the allele APOE E4, the short allele D19S178 and the long allele APO CII.
5. Use according to Claim 4, characterised in that the short allele D19S178 comprises less than 167±4, in particular less than 167 nucleotides.
6. Use according to Claim 4, characterised in that the long allele APO CII comprises more than 30±3, in particular more than 30 repetitive patterns (CA).
7. Method of diagnosis for Alzheimer's disease, characterised in that the presence of at least two of the following markers is sought in a biological sample of a patient: allele APOE E4, the short allele D19S178 and the long allele APO CII.
8. Method according to Claim 7, characterised by the following steps:

   a) placing the biological sample containing DNA in contact with a pair of specific activators allowing the amplification of all or part of the genes APOE, D19S178 and/or APO CII, the human DNA contained in the sample having possibly been made accessible to hybridisation and in conditions allowing hybridisation of the activators to the human DNA contained in the biological sample;

   b) amplification of the human DNA;

   c) disclosure of products of amplification by appropriate techniques;

   d) detection of the possible presence of the alleles APOE ε4, short D19S178 and long APO CII by appropriate techniques.
9. Method according to Claim 8, characterised in that the presence of the alleles APOE ε4, short D19S178 and long APO CII is proven by the technique of electrophoresis on polyacrylamide gel.
10. Kit for conducting a method according to one of Claims 8 to 10, characterised in that it comprises:

    pairs of specific activators of at least two of the genes APOE, D19S178 and APO CII,

    the necessary reagents for conducting the amplification of DNA,

    possibly reagents allowing the detection of the alleles APOE ε4, short D19S178 and/or APO CII, for example necessary reagents for electrophoresis on polyacrylamide gel and the disclosure of fragments after migration,

    possibly standards of reference formed by wild alleles of mutant genes.
11. Products of amplification of all or part of at least two genes selected from APOE, APO CII and D19S178.
12. Diagnostic composition formed from at least two genetic markers selected from APOE, APO CII and D19S178.